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Back to Journal »International Journal of Nanomedicine» Volume 13

Author Islami M, Zarrabi A, Tada S, Kawamoto M, Isoshima T, Ito Y 

Published on October 5, 2018, Volume 2018: 13 pages 6059—6071

DOI https://doi.org/10.2147/IJN.S178374

Single anonymous peer review

Editor approved for publication: Dr. Thomas Webster

Matin Islami,1 Ali Zarrabi,1 Seiichi Tada,2 Masuki Kawamoto,2,3 Takashi Isoshima,3 Yoshihiro Ito2,3 1 Department of Biotechnology, Faculty of Advanced Science and Technology, Isfahan University, Isfahan 8174674441, Iran; 2 Emerging Bioengineering Materials Research Team, RIKEN Emerging Material Science Center, Wako, Saitama 351-0198, Japan; 3 Nano Medical Engineering Laboratory, RIKEN Pioneer Research Cluster, Wako, Saitama 351-0198, Japan Purpose: Based on graphene oxide, use The simple and one-step strategy to control the release of anticancer drugs creates an efficient drug delivery system. Method: The single-layer graphene oxide (GO) sheet was manufactured through an improved and improved Hummers method. The biocompatible hyperbranched polyglycerol (HPG) was grafted on the surface of GO through the ring-opening hyperbranched polymerization of glycidol. Different ratios of GO and glycidol are used for polymer grafting. The anticancer drug quercetin (Qu) was loaded into the modified GO through non-covalent interactions. Results: The results obtained by Fourier transform infrared spectroscopy and Raman spectroscopy, thermogravimetric analysis, energy dispersive X-ray and X-ray spectroscopy, scanning electron microscope and atomic force microscope confirmed the polymer grafting on the surface of the GO sheet. The results show that the aggregation increases the d-spacing between the basal planes. In addition, as a hydrophilic polymer, HPG improves the stability and dispersibility of GO tablets in biological solutions, and gives the tablets additional drug-carrying capacity. By comparing the drug loading and release of HPG-modified GO and linear PPO-modified GO, the effect of hyperbranched structure on drug loading and release was studied. Our experiments show that HPG-GO has high drug loading (up to 185%) and excellent encapsulation efficiency (up to 93%) compared with linear PO grafted GO. The release profile of Qu at different pH levels showed controlled and sustained drug release. HPG-GO has no initial burst effect, indicating that acidic solutions can promote drug release. During the 72-hour incubation process, HPG-GO did not show any cytotoxicity to MCF7 cell lines at different concentrations. The uptake and entry of HPG-GO into cells was verified by measuring the amount of Qu in cells by high performance liquid chromatography. Conclusion: The unique properties of GO, combined with the biodegradable polymer polyglycerol, show high drug loading capacity, pH-dependent drug release, and cell compatibility with HPG-GO, making it a promising Nanocarriers are used for anti-cancer drug delivery. Keywords: polyglycerol, graphene oxide, hyperbranched polymer, cancer cell

There is an urgent need to develop biocompatible drug carriers to improve the therapeutic effect of hydrophobic anticancer drugs. Nanomaterial-based drug delivery systems can improve the efficiency of free hydrophobic drugs by reducing systemic toxicity and increasing drug solubility and cellular uptake. High drug loading and triggering drug release near cancer cells are two important issues for the introduction of nanocarriers as a suitable drug delivery system. Due to its amazing and unique physical and chemical properties, graphene derivatives have attracted great attention in biomedical applications. Graphene sheet is a single layer of sp2 hybridized carbon atoms arranged into a two-dimensional honeycomb lattice 1-4 and a new type of matrix biomaterial. It appears in the 2004.5 polar solvent through van der Waals interaction and π-π to form irreversible graphene aggregates Stacking limits its biomedical applications. 6

Graphene oxide (GO) is one of the most important derivatives of graphene. It has hydroxyl and epoxy groups on the base surface, and carboxyl groups 4, 7 on the edges, which provide better dispersibility in pure water and Provide reaction sites for further surface chemistry8,9 Unlike graphene sheets, polar groups can promote the water solubility of GO nanosheets,10 making it widely used in the biomedical field, including biosensing, molecular imaging, Gene transfection and drug delivery. 4,11-15 GO’s characteristics, such as high aspect ratio, large specific surface area, rich surface chemistry and good dispersibility in aqueous solutions, significantly improve its loading efficiency of aromatic molecules (such as anti-cancer drugs), and It was introduced as a more effective method. Drug delivery systems 11, 16, 17 are better than other carbon nanomaterials 18. These are the main driving forces for the development of the GO drug delivery system in the past decade. Although GO forms a stable suspension in pure water, 19 it has strong π-π accumulation and van der Waals interactions through strong π-π accumulation in solutions rich in salt or protein (such as cell culture medium and serum). The tendency to form irreversible agglomerates into multilayer GO, 19 this poses a practical challenge for GO biomedical applications.

In recent years, in order to optimize the biocompatibility, solubility and stability of GO tablets in physiological media through non-covalent or covalent functionalization, many experiments have been conducted. 11, 16, 20-22 This functionalization improves the stability and biocompatibility of GO in physiological media. Suppress the water phase by preventing the formation of van der Waals forces and π-π interactions. Chemical modification of carbon-based nanomaterials with polymers seems to be an effective method. 23 Dai et al. 8 first used a six-arm PEG-GO complex as a new nanocarrier to load water-insoluble anticancer drugs through hydrophobicity and π. -Π Superposition interaction. Polymers such as chitosan, 17,24 polyvinyl alcohol, 25 and poly-L-lysine 21 have been shown to increase the water solubility of graphene and its derivatives. The biocompatible coating of GO can transform it into a promising biomaterial, which has great potential for controlled anticancer drug delivery. Aliphatic polyether polyol polyglycerol (PG) is known as a hyperbranched polymer with a degree of branching as high as 0.52–0.59.26. Nanostructures based on hyperbranched PG (HPG) have been extensively studied due to its biodegradability. Applicable carrier in biotechnology and pharmacology, high biocompatibility and excellent solubility in aqueous media. 27-32

In this work, based on the in-situ ring-opening polymerization of GO, an efficient GO-based drug delivery system was designed by covalently grafting HPG to the surface of GO for the controlled release of quercetin (Qu), A hydrophobic anti-cancer drug. Graft glycidol and polymer through a one-step strategy. As a hyperbranched polymer, PG can expand the distance between GO nanosheets, so that drug molecules can be physically embedded in the cavities between the polymer branches, which is conducive to achieving greater drug loading. The interaction between HPG-GO and drug molecules is based on several types of interactions, such as hydrophobic interactions, hydrogen bonding, and π-π stacking. 33 In addition, the drug-carrying capacity and drug release profile of HPG-GO were grafted with GO with linear polypropylene oxide (PPO). The conclusion is that combining the inherent properties of GO with biodegradable PG can produce promising drug delivery systems.

Qu came from Sigma-Aldrich (St. Louis, Missouri, USA). Graphite powder (77882-42-5; Merck, Darmstadt, Germany) is used for GO preparation. PO 99% was purchased from Tokyo Chemical Industry, and 95% glycidol was purchased from Kanto Chemical (Tokyo, Japan). Sulfuric acid (H2SO4) 95%, phosphoric acid (H3PO4) 95%, nitric acid (HNO3) 65%, hydrogen peroxide 30%, hydrochloric acid (HCl) 37%, ethanol (≥99.5%), acetone, anhydrous dimethyl sulfoxide (DMSO) , 0.1% Triton X-100 and 99.7% 2-propanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Methanol 99.8% was purchased from Junsei. Potassium permanganate (KMnO4) was purchased from Merck. MCF7 and L929 cells were purchased from JCRB cell bank (Osaka, Japan). DMEM, FBS, and 1% penicillin-streptomycin were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The WST-8-based cell counting kit (CCK8) and dialysis bags (molecular weight 1,000 and 15,000 Da) were purchased from Dojindo (Kumamoto, Japan) and Repligen (Waltham, MA, USA), respectively. All other reagents are of analytical grade and used as is.

Two methods are used to oxidize graphite flakes: the modified 34 and the modified Hummers method.

Synthesize GO with the improved Hummers method

In the first step, 0.5 g (1 weight equivalent) of graphite flakes were pretreated with a 3:1 volume ratio of concentrated H2SO4:HNO3 at room temperature for 24 hours. Then deionized (DI) water (120 mL) was added to the resulting solution to quench the reaction. The resulting product was washed with deionized water and freeze-dried. In the second step, 0.5 g of the product was mixed with 110 mL H2SO4, and 1.3 g (3 weight equivalents) potassium permanganate (KMnO4) was gradually added in an ice-water bath within 1 hour to keep the reaction temperature below 20°C The resulting solution was then stirred at room temperature for 24 hours. After that, 300 mL of deionized water was added to the mixture to produce a highly exothermic reaction. The temperature of the solution reached 25°C after 30 minutes. The reaction was terminated by adding a 3:1 H2O2 aqueous solution with a volume ratio of 3:1, resulting in a yellow-brown mixture. The mixture was then centrifuged (10 minutes, 4,000 rpm) and washed twice with HCl solution (37%) and three times with deionized water.

Synthesize GO with the improved Hummers method

Adding a 9:1 mixture of concentrated H2SO4/H3PO4 to graphite flakes (1 gram, 1 weight equivalent) and KMnO4 (6 grams, 6 weight equivalent) produces a slight exothermic reaction (35°C–40° C). The reaction temperature was raised to 50°C and the mixture was stirred for 12 hours. The reaction was cooled to room temperature and poured onto ice (100 mL) containing H 2 O 2 (1 mL). Then centrifuge (10 minutes, 4,000 rpm) and wash twice with HCl (37%) solution and three times with deionized water. In these two processes, NaHCO3 is used for neutralization. GO uses a dialysis bag (14,000 Da cut-off value) to dialyze in deionized water for 48 hours to remove impurities. The dialysis product was centrifuged and freeze-dried to produce GO powder. This procedure is followed by sonication to produce GO nanosheets.

Polymer grafted to graphene oxide

Different predetermined amounts of glycidol (Table 1) and GO powder were mixed and sonicated in an ice water bath for 2 hours. Subsequently, the mixture was magnetically stirred in an oil bath at 140°C for 20 hours under a nitrogen atmosphere. The resulting black gel-like product was cooled and dissolved in methanol, and the resulting mixture was precipitated in acetone (10 minutes, 4,000 rpm). Finally, HPG-GO was dialyzed against a dialysis bag (1,000 Da cut-off value) in deionized water for 24 hours to remove solvents and impurities. After purification, the product was centrifuged and freeze-dried.

Table 1 Summary of polymer grafting to graphene oxide. Abbreviations: Gly, glycidol; PO, propylene oxide.

The GO and PO were sonicated in an ice water bath for 2 hours, and then magnetically stirred at 40°C for 20 hours to initiate the polymerization of PO on the GO surface. The linear PPO grafted deposit is obtained by a method similar to that of HPG-GO.

Thermogravimetric analysis (TGA) uses the STA 503 analyzer to increase the temperature from ambient to 800°C at a heating rate of 10°C/min in an argon atmosphere. Fourier transform infrared (FTIR) spectra were recorded on an IR Prestige 21 spectrometer (Shimadzu Corporation, Kyoto, Japan) at 400–4,000 cm-1 with a resolution of 1 cm-1, using potassium bromide particles. Raman spectroscopy was performed on NRS-5100 (Jasco, Tokyo, Japan) using an excitation laser with a wavelength of 532 nm. A SmartLab® goniometer (Rigaku Corporation, Tokyo, Japan) was used to collect 2°–65° X-ray diffraction (XRD) patterns at a scan rate of 2°/min. SmartLab (Rigaku Corporation) was also used to generate 1°-3° XRD patterns using CuKa radiation (λ=1.5406 Å). The Atomic Force Microscope (AFM) image was obtained with an MFP-3D microscope (Oxford Instruments, Abingdon, UK) in AC mode using an NCH probe (NanoWorld, Neuchâtel, Switzerland) to measure thickness and particle size distribution. The AFM sample was prepared by spin-coating an aqueous solution of nanosheets onto a freshly cut silicon substrate. Morphological and elemental analysis were obtained using a field emission scanning electron microscope equipped with an energy dispersive X-ray (EDX) spectrometer (MIRA3; TESCAN, Brno, Czech Republic). Ultraviolet (UV)-visible light spectrophotometry (V-550; Jasco) was used to measure the absorption spectrum.

Load songs in HPG-GO and PPO-GO

The drug loading behavior of HPG-GO nanocarriers was evaluated by encapsulating Qu as a drug model. Disperse HPG-GO in deionized water for 1 hour to produce a homogeneous solution. Mix different ratios of nanocarrier aqueous solution and Qu dissolved in ethanol (ratio of 1:1, 1:2, and 2:1) and shake vigorously at 4°C for 12, 18, and 24 hours. The product (drug-loaded nanocarrier) was collected by centrifugation. The amount of free Qu is determined by the UV visibility at 373 nm. In order to quantify free Qu, a standard curve was prepared by measuring the UV absorbance of a series of Qu solutions of known concentration in ethanol. Qu load capacity and retention efficiency are calculated according to equations 1 and 2:

In addition, according to the same protocol, the load capacity of PPO-GO was compared with that of HPG-GO.

Release Qu from HPG-GO and PPO-GO nanocarriers

The drug release curves of HPG-GO and PPO-GO nanocarriers were studied under physiological temperature of 37°C, pH 6.6 (environmental pH of the tumor) and pH 7.4 (physiological pH). In short, transfer 2 mL of the drug-loaded nanocarrier aqueous solution to a dialysis bag (1,000 Da cut-off), immerse it in 25 mL PBS 0.1 M and incubate at 37°C. All drug release media are withdrawn after a certain time interval. Use fresh medium for each measurement. The amount of Qu released into the buffer solution was measured using ultraviolet-visible spectrophotometry. Calculate the cumulative release (%) of drug-loaded nanocarriers according to Equation 3:

Human breast cancer MCF7 cell line and normal mouse fibroblast L929 cell line were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells are cultured in a humid environment at 37°C containing 5% CO2.

The WST8 assay was performed to evaluate the cytotoxicity of free Qu and GO, HPG-GO and Qu-HPG-GO nanocarriers. Qu was dissolved in DMSO (1%) and diluted in DMEM to evaluate the growth inhibition of MCF7 cells. The cells were seeded in a 96-well plate at a density of 104 cells/well. Then, the cells are incubated with samples of different concentrations for a predetermined time. The relative cell viability was assessed by adding CCK8 (10 μL/well) after 2 hours. A fluorescence microplate reader (PerkinElmer Inc., Waltham, MA, USA) was used to quantify the cytotoxic activity of the compounds at 450 nm. Data are expressed as mean ± SD.

MCF7 and L929 cells (2.5×105 cells/well) were respectively seeded in a 24-well plate and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, after 24 hours, the medium was changed to a fresh medium containing free Qu (dissolved in 1% DMSO) and diluted Qu-HPG-GO nanocarriers at a concentration of 50 μg/mL. Absorption studies are conducted in a time-dependent manner. At predetermined time intervals, remove the medium and wash the cells twice with cold PBS (pH 7.4). In addition, the cells were lysed with 0.1% Triton X-100, and then the internalized drug was solubilized by washing the cells with methanol. The cell extract was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was analyzed by high performance liquid chromatography (HPLC) to quantify the drug. A Smartline 2600 HPLC system (KNAUER, Berlin, Germany) equipped with a ProntoSil C18 (5 μm–150 mm) column and UV detector (detection wavelength at 373 nm) was used for sample analysis. The mobile phase used for the analysis is a mixture of methanol and water (0.1% orthophosphoric acid, 35:65 v:v). The flow rate was maintained at 1 mL/min, 20 μL was injected, the running time was 30 minutes, and the column temperature was 25°C. The concentration of Qu is determined by comparing the peak area with the standard curve.

In order to obtain GO, graphite exfoliation usually occurs under the action of strong oxidants. The mixture of KMnO4 and NaNO3 in concentrated sulfuric acid and the mixture of KMnO4 and concentrated H2SO4 can be regarded as common oxidants. The specific oxidant, the source of graphite and the reaction conditions have a great influence on the final product. 35 We use flake graphite as the mineral source of graphite to synthesize GO. These procedures will not completely preserve the structure of the graphene sheet, and significant defects will appear in the sheet structure. 36 Recently, Marcano et al. have demonstrated that treating graphite with H3PO4 can keep the graphite base surface more complete. 34

For the sake of clarity, the term "IGO" is used herein to refer to products with improved methods, and "MGO" is used to refer to products with improved methods. For the improved Hummers method, in addition to KMnO4, HNO3 also acts as an oxidant and is introduced to react strongly with the aromatic carbon surface. 37 Graphite oxidation provides GO with various oxygen-containing substances, such as epoxides, alcohols, ketone carbonyl compounds, and carboxyl groups, in its structural lattice. 34

The FTIR spectrum of graphite oxide did not show any obvious absorption peaks (Figure 1). The FTIR spectrum of GO revealed the existence of characteristic peaks: OH stretching vibration (3,420 cm-1), C=O stretching vibration (1,724 cm-1), C=C (1,616 cm-1) from the unoxidized sp2 CC bond, The COH plane bending vibration (1,382 cm-1) in the epoxy group, the COC stretching vibration (1,253 cm-1) and the CO stretching vibration in the alkoxy group (1,064 cm-1) verify the formation of carboxyl and hydroxyl groups. The oxidation of flake graphite increases water solubility and surface functional groups (carboxyl and hydroxyl) that can be used for subsequent polymer grafting. Regarding FTIR spectroscopy, compared with IGO wafers, MGO wafers have fewer basal defects.

Figure 1 FTIR spectra of GO and HPG-GO nanocarriers. Abbreviations: FTIR, Fourier transform infrared; GO, graphene oxide; HPG, hyperbranched polyglycerol; MOD, modified Hummer; IMP, improved Hummer method.

Raman spectra show great similarity in IGO and MGO sheets. Two prominent characteristic peaks can be seen in the Raman spectrum: the D bond at ~1,350 cm-1 and the G bond at ~1,590 cm-1, confirming the lattice distortion (Figure 2A). The G band is attributed to the domain of sp2 hybridized carbon, and the D band is attributed to structural defects. The intensity ratio of the D band to the G band (ID:IG) is considered to be an indicator of the degree of disorder in graphene-based materials. The recorded GO film spectrum shows that the ID:IG ratios of IGO and MGO are 0.98 and 0.89, respectively. Raman spectroscopy shows that the structure of the MGO sheet is more regular than that of IGO, indicating that more basal planes are retained.

Figure 2 Raman spectrum (A); ID:IG ratio (B) of GO and HPG-GO nanocarriers are recorded. Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol.

The XRD spectra of the original graphite and GO powder samples are shown in Figure 3. The strong and sharp peak corresponding to the (002) plane of graphite appears at 2θ=26.5°, while the peak of GO shows a sharp diffraction peak (001) =10.4° at 2θ, which corresponds to the presence of oxygen-containing functional groups. The oxidation process is formed. 38 The interlayer spacing of the material is directly proportional to the degree of oxidation. The functional group causes the d-spacing to increase from 0.33 nm (2θ=26.5°) to 0.85 nm (2θ=10.4°). The same overall oxidation sequence can be derived from the XRD diagram. The increase in the interlayer spacing from 0.33 nm for graphite to 0.85 nm for MGO and IGO sheets is attributed to the destruction of the carbon lattice, rather than the continuous aromatic lattice of graphene.

Figure 3 XRD spectra of graphite, GO and HPG-GO nanocarriers recorded in different ranges: 1°–3° and 3°–50°. Abbreviations: XRD, X-ray diffraction; GO, graphene oxide; HPG, hyperbranched polyglycerol.

Polymer grafted to GO sheet

Functional groups provide the possibility of surface modification for GO sheets. The hydroxyl and carboxyl groups on the surface of GO initiate the polymerization of epoxy ring-opening branching. As a substrate, GO increases the order and level of polymerization. The polymerization of the monomer and the grafting of the resulting polymer on the surface of the GO sheet occur simultaneously. The successful polymerization of glycidol and the grafting of the polymer on the surface of the GO sheet were carried out through different ratios of glycidol and GO powder (Table 1).

FTIR spectroscopy verified the covalent attachment of PG on the surface of two types of GO flakes (M and I) (Figure 1). The FTIR spectrum of the HPG-GO nanocarrier is different from the FTIR spectrum of the GO sheet because there are new pickups at ~2,900 and ~1,240 cm-1, which are attributed to the stretching vibrations of the methylene CH and CO-C bonds located respectively Epoxy group. This supports the grafting of HPG on the surface of the GO sheet. ID:IG indicates the degree of disorder of graphene-based materials. Raman spectroscopy shows that the ID:IG ratio of modified GO materials increases in the order of MGO <HPG-IGO ≤ IGO <HPG-MGO. Based on these findings, it is recommended to use MGO instead of IGO for polymer grafting. In addition, in the case of HPG-MGO, the use of 1 mL and 1.5 mL monomer resulted in an increase in the ID:IG ratio of 1.04 and 1.20, respectively. However, a further increase in monomer reduced the ID:IG ratio to 1.10 (Figure 2B). The upward trend of ID:IG is related to the decrease in the average size of sp2 domains and a higher degree of disordered structure, which is due to the polymer grafting process.

XRD was also used to study the crystallinity of the synthesized modified GO (Figure 3). The diffraction peak at 2θ=2° indicates that HPG was successfully grafted on the surface of GO. Compared with GO, HPG-GO sheet exhibits a larger d-spacing than GO, which is attributed to polymer grafting on the GO surface (Table 2). In addition, the broad reflection peak appearing at ~20° is related to grafted PG as a polyepoxide, confirming our hypothesis. According to Raman spectroscopy, XRD patterns indicate that MGO sheets are more suitable substrates for polymer grafting than IGOs. By increasing the concentration of glycidol, the ID:IG ratio and d-spacing were initially increased more than pure GO sheets. However, when more monomers are added, these factors will decrease, and eventually a higher concentration of monomers will cause the ID:IG ratio and d spacing to increase again. This phenomenon may be due to polymerization and branching that occur at low concentrations. As more monomers are added, the branch returns to the inside. In particular, the terminal groups can be folded back into the molecule through hydrogen bonds, 39,40 and finally after the internal space is saturated, the branches will re-grow to the outside at a higher monomer concentration. Use AFM to characterize the thickness and size of GO and HPG-GO sheets. As shown in Figure 4A, the thickness and diameter of a single GO sheet are <1 nm and ~0.3-0.7 μm, respectively. Considering the typical thickness of single-layer GO flakes (approximately 0.71 nm),3,41 most of the synthesized GO flakes are single-layered.

Table 2 Summary of d-spacing of nanosheets (nm) Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol.

Figure 4 AFM of GO flake (A), HPG-GO flake (B), HPG-GO oval (C). Abbreviations: AFM, atomic force microscope; GO, graphene oxide; HPG, hyperbranched polyglycerol.

The AFM image shows the changes in the thickness of the HPG-GO sheet: 3.5 and 7 nm related to single and double layers, respectively (Figure 4B). Statistical analysis shows that the thickness of HPG-GO sheet has increased compared with the original GO due to polymer grafting. It is worth noting that the lateral size of some flakes is ~0.26 μm smaller than that of the original GO flakes (Figure 4C). Observations of the elliptical HPG-GO sheet showed that the polymer condensation reaction process resulted in a reduction in size. 7

The field emission scanning electron microscope provides morphological information of GO sheets and HPG-GO nanocarriers (Figure 5). The GO flakes are flakes with sharp edges and flat surfaces. In contrast, HPG-GO sheets show a thicker and wrinkled surface. This proves that the polymer was grafted onto the surface of the GO sheet. The wrinkled surface not only provides a high surface area for more drug loads, but also prevents the aggregation of GO flakes. 17 EDX analysis is used to characterize the chemical composition of GO and HPG to support FTIR results. In addition, an EDX map (element distribution image) is provided to show a meaningful picture of the element distribution on the surface, and to verify the presence of carbon and oxygen elements on the entire surface of the GO and HPG-GO sheets (Figure 6). The EDX spectrum also shows the carbon (C) and oxygen (O) peaks of GO and HPG-GO sheets. Due to the removal of physically adsorbed water, the TGA curve of GO shows the first stage of weightlessness below 100°C. The second stage, which occurred around 180°C–220°C, was attributed to the pyrolysis of the oxygen-containing groups on the GO sheet,1 and the overall GO lost nearly 50% of its total weight (Figure 7). In contrast, the TGA curve of HPG-GO showed a major weight loss in a wider temperature range of 300°C–420°C, which was attributed to the thermal degradation of PG grafted on the surface of the GO sheet. 1 The content of grafted polymer is estimated to be about 70% by weight. The increase in the amount of monomer leads to a slight increase in the polymer content.

Figure 5 SEM of GO sheet (A) and HPG-GO sheet (B). Abbreviations: SEM, scanning electron microscope; GO, graphene oxide; HPG, hyperbranched polyglycerol.

Figure 6 EDX spectra of GO flakes (blue) and HPG-GO flakes (red). Abbreviations: EDX, energy dispersive X-ray; GO, graphene oxide; HPG, hyperbranched polyglycerol.

Figure 7 shows the TGA data recorded by GO and HPG-GO nanocarriers. Abbreviations: TGA, thermogravimetric analysis; GO, graphene oxide; HPG, hyperbranched polyglycerol.

Drug loading and encapsulation efficiency

Phytochemicals, such as polyphenols and carotenoids, have received great attention due to their contribution to human health. 42-44 It has been reported that Qu has a variety of biological activities, such as anti-cancer, anti-oxidation and anti-inflammatory. As a polyphenol, Qu is the main representative of the flavonoid subclass, which limits the negative effects of free radicals by quickly transferring hydrogen atoms to free radicals. 44,45 However, low water solubility and instability in physiological media hinder its application of compounds, but this therapeutic molecule can be embedded in biodegradable polymer nanoparticles as a way to improve the efficacy of Qu method. Nanosystems can protect chemically unstable biologically active compounds in target pathways and blood circulation, and can also enhance the water solubility, biological half-life and subsequent bioavailability of hydrophobic drugs, thereby enhancing the therapeutic effect of Qu and improving biodistribution.

The drug loading was determined at different time intervals and various ratios of nanocarriers and drugs. Although our data indicate that the ratio of nanocarriers and drugs has a significant effect on this ability, increasing the exposure time does not affect the drug loading ability. The Qu concentration present in different samples is obtained by standard regression, based on equation 4:

Table 3 and Table 4 respectively summarize the average drug loading and encapsulation efficiency of HPG-GO achieved by the optimized formula. Our observations show that Qu-loaded HPG-GO has the highest encapsulation efficiency (93%) and the highest drug loading (185.4%). By increasing the amount of monomer, the encapsulation efficiency and drug loading of Qu increased from 40% to 93% and 80% to 185.4%, respectively. This can be attributed to the saturation of the hyperbranched polymer structure. In addition, we compared the drug-carrying capacity of HPG-GO and linear polymer grafted GO (PPO-GO). The drug loading of hyperbranched polymers is approximately five times that of linear polymers.

Table 3 Summary of drug loading (%) Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol.

Table 4 Summary of drug encapsulation efficiency Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol.

The release behavior of polymer-GO nanocarriers was recorded at different pH values ​​(Figure 8). In the first 12 hours, HPG-GO nanocarriers released 20.6% and 27% Qu in natural and acidic pH PBS, respectively. After 72 hours, the total drug released rose to 36.5% and 49.2% at natural and acidic pH values, respectively. The different release rate at 72 hours may be related to the different positions of the drug in the nanocarrier.

Figure 8 Cumulative quercetin release (%) of HPG-GO and PPO-GO nanocarriers at 37°C in PBS (pH 6.6 and 7.4) at different time points. Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol; PPO, polypropylene oxide.

The in vitro release test showed that the release rate of HPG-GO nanocarriers under acidic pH was higher than that of natural pH. The drug release behavior of PPO-GO nanocarriers was also studied under the same conditions. Qu is released rapidly in the initial stage: 40.2% of the loaded drug is released after 12 hours of incubation at pH 7.4, and the maximum release rate reaches 92% after 72 hours. However, 31.6% of the drug was released during the same incubation time at pH 6.6 and increased to 80% after 72 hours. Under the same physiological conditions, the drug release curves of HPG-GO and GO-PPO nanocarriers are significantly different.

Compared with PPO-GO nanocarriers, HPG-GO nanocarriers exhibit sustained and slow release, avoiding the initial burst release of Qu. The controlled and sustained release of HPG-GO nanocarriers may be due to the retention of the drug in the cavity, the hydrophobic attraction between the hydrophobic segments of the polymer, the hydrophobic bioactive compound 46, and the hydrogen bond between the polymer and the drug. It may reduce the diffusion of Qu. On the other hand, the hydrogen bonds and hydrophobic interactions between hyperbranched polymers and drugs are stronger than linear polymers and drugs and drugs, because there are a large number of hydroxyl groups and hydrophobic cavities inside the structure of hyperbranched polymers. In addition, it has also been found that these graphene derivatives exhibit a drug release profile that depends on the pH value. Based on the drug release mode of HPG-GO nanocarriers, it shows that acidic solutions can promote drug release. The Qu release rate of HPG-GO nanocarriers was faster at pH 6.6 than at pH 7.4. This can be attributed to the hydrogen bond between the drug and HPG, which is more prominent under neutral conditions, leading to a sustained release mode. The hyperbranched three-dimensional structure is significantly affected by the polarity of the solvent. 39 The interaction between water and polymer takes precedence over the weak interaction between polymer and Qu. 47 Compared with acidic solutions, the release rate at neutral pH is higher, and the rapid burst effect is not suitable for anti-cancer drug delivery and cancer treatment, because the macrophages of the reticuloendothelial system will immediately remove the drug from the cancer before reaching the cancer tissue. Cleared in blood circulation (pH 7.4). Due to the acidic tumor environment, the pH-dependent drug release of HPG-GO nanocarriers can be used for controlled drug delivery applications, which subsequently reduces premature drug release and its related side effects. HPG seems to play two key roles in HPG-GO nanocarriers: stabilizing the GO sheet in the electrolyte solution and preventing the rapid release of the drug in a neutral medium. It may be a good candidate for the release of smart drugs.

The cell compatibility of nanocarriers is a prerequisite for the application of drug delivery systems. The WST8 assay was used to evaluate the concentration and time-dependent cytotoxicity of free drugs, GO, HPG-GO, and Qu-HPG-GO nanocarriers on MCF7 cells. As shown in Figure 9, HPG-GO tablets did not affect cell viability at any incubation time or study concentration, while GO, Qu-HPG-GO and free Qu induced concentration and time-dependent toxicity to MCF7 cells. After treatment at 25 and 50 μg/mL for 24 hours, GO did not show any significant changes in cell viability, while 100 μg/mL reduced cell viability by 96%. It is worth noting that for all the studied concentrations, cell viability decreased after 48 hours and 72 hours. These results indicate that the cytotoxicity of GO is directly related to time and concentration. According to the drug release rate over time, loading Qu in HPG-GO nanocarriers resulted in decreased cell viability and increased the concentration of Qu in HPG-GO nanocarriers. According to the chart, free song showed obvious toxic effects on cells. The best concentration (100 μm/mL) of free Qu has IC50 values ​​of 95, 123/76 and 135/13 after 72, 48 and 24 hours, respectively.

Figure 9 Relative survival rate of MCF7 cells incubated with quercetin preparations at different incubation times. Note: Measured at 24 hours (A), 48 hours (B) and 72 hours (C) by standard WST8. Each data point shows the mean ± SD (n=3).

Compared with free Qu, the IC50 values ​​of Qu-HPG-GO nanocarriers were calculated to be 137, 203/25 and 943/39, respectively. Compared with free Qu, the lower cell killing ability of Qu-HPG-GO nanocarriers may be attributed to the delayed release of Qu, because only 32.92%, 41.72% and 49.22% of the drugs are released after 24, 48 and 72 hours3 Only 50% of the drug is released after days. The chart shows that as the concentration of free drugs or drug-loaded nanocarriers increases, the viability of MCF7 cells decreases significantly. Compared with free Qu, HPG-GO nanocarriers enhanced the solubility of Qu in cell culture media.

The cell uptake and intracellular amount of free Qu and Qu loaded in HPG-GO nanocarriers of normal cells and cancer cells were studied by HPLC. Figure 10 depicts the quantitative cellular uptake of MCF7 and L929 cells. After 12 hours of incubation, the Qu uptake of drug-loaded HPG-GO and free Qu of the two cell lines was almost similar (approximately 27%), and after 24 hours of incubation, the changes in Qu levels were negligible compared with free Qu. Exposure to Qu-HPG-GO. This phenomenon can be attributed to two factors. First, according to the toxicity results, only 33% and 25% of the drug was released after 24 hours under acidic and neutral conditions, respectively. Therefore, only one-third of the drug was released after 24 hours. Second, the different pathways of cellular uptake will affect the rate of drug release. As we all know, endocytosis is a way for nanocarriers to be internalized into cells, and free Qu molecules are transported into cells through passive diffusion. 48-50 It is worth noting that Qu is very hydrophobic and does not dissolve in water or cell culture media. Therefore, DMSO (1%) is used as an adjuvant to increase the solubility of Qu. In our body, the solubility of free koji is very low and the bioavailability is very low.

Figure 10 Time-dependent uptake of Qu preparation by MCF7 cells. Abbreviations: GO, graphene oxide; HPG, hyperbranched polyglycerol.

In summary, we synthesized GO using an improved and improved Hummers method. Based on the in-situ ring-opening polymerization of glycidol, biocompatible HPG was grafted onto the surface of GO through a one-step procedure, thereby developing HPG-GO nanocarriers. Compared with IGO, MGO achieves a more ideal polymer grafting. The increase in d-spacing of HPG-MGO was observed only when the amount of monomer used for polymerization increased. This effect can provide more interlayer space for increasing the drug loading. The thicker and wrinkled surface of the polymer grafted with HPG-MGO sheet was introduced as a suitable nanocarrier for the delivery of the hydrophobic aromatic anticancer drug Qu. In addition, it was found that the drug release of HPG-GO nanocarriers was more durable and controllable than PPO-GO (as linear nanocarriers). The WST8 assay was used to verify the excellent cell compatibility of HPG-GO nanocarriers, indicating that Qu-HPG-GO induces the inhibition of MCF7 cells in a concentration and time-dependent manner. Our in vitro quantitative cell study of Qu-HPG-GO nanocarriers shows that surface functionalization plays an important role in improving biocompatibility and the full absorption of graphene-based nanomaterials. This study points out that HPG-GO nanocarriers are a promising and effective cancer therapy drug delivery system.

We want to confirm that there are no known conflicts of interest related to this publication, and we have not provided any significant financial support for this work that may affect its results.

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